Chromatin immunoprecipitation assay. The E2F1 and protein complexes were cross-linked inside the SaOS-2
Daunorubicin HCl by the addition of formaldehyde (1% final concentration) to the cells in culture. Whole cell extracts were prepared using sonication and an aliquot of the cross-linked protein complexes were immunoprecipitated by incubation with either the E2F1 specific antibody (Santa Cruz Biotechnologies, CA) or IgG antibody overnight at 4 °C with rotation. Chromatin-antibody complexes were isolated from solution by incubation with protein G-Sepharose beads for 1 h at 4 °C with rotation. The Sepharose-bound immune complexes were washed and eluted from beads with elution buffer (1% SDS and 0.1 M NaHCO3). Elute was heated at 65 °C for 4 h to reverse the formaldehyde cross-linking and DNA extracted. DNA samples from
chromatin immunoprecipitation preparations were analyzed by PCR using primers spanning RANKL gene in the region of promoter (Forward, 5′-CTTGGAGGGCCCCCATGAAGGA, Reverse, 5′-CCCTTCTTGTCTGCGGCCAACT).
